|
| Status |
Public on Dec 07, 2010 |
| Title |
Hum_Mammary_media_Old_M104 |
| Sample type |
RNA |
| |
|
| Source name |
Media from human mammary artery, patient over 75 (old)
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
age: 75
tissue: Mammary artery
surgical procedure: coronary bypass surgery (surgical waste)
|
| Biomaterial provider |
Surgical department of the cardiology institute at Hôpital Pitié-Salpêtrière, Paris, France
|
| Treatment protocol |
The mammary artery segments was kept in physiological solution at 4°C and, the same day,was dissected by mechanically dissociating the media from the adventitia and immediately freezing it in liquid nitrogen.
Frozen medias (usually 50-150 mg) were crushed in ice in 6 volumes of Lysis Binding Buffer from mirVana miRNA isolation kit (Ambion, Applied Biosystems, Courtaboeuf, France) and total RNA were prepared according to the manufacturer’s instructions. 5-20 µg total RNA were obtained.
To avoid any genomic contamination, each RNA was treated with rDNase I using DNA-freeTM DNase treatment and removal reagents from Ambion (Applied Biosystems, Courtaboeuf, France).
RNA concentrations were determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Courtaboeuf, France). RNA quality was assayed using RNA nano LabChip ® and a 2100 Bioanalyzer (Agilent Technologies, Massy, France). Only patients with a RNA Integrity number greater than 7 (determined by the Bioanalyzer) and a 28S/18S value greater than 1 were selected for the following studies.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen medias (usually 50-150 mg) were crushed in ice in 6 volumes of Lysis Binding Buffer from mirVana miRNA isolation kit (Ambion, Applied Biosystems, Courtaboeuf, France) and total RNA were prepared according to the manufacturer’s instructions. 5-20 µg total RNA were obtained.
To avoid any genomic contamination, each RNA was treated with rDNase I using DNA-freeTM DNase treatment and removal reagents from Ambion (Applied Biosystems, Courtaboeuf, France).
RNA concentrations were determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Courtaboeuf, France). RNA quality was assayed using RNA nano LabChip ® and a 2100 Bioanalyzer (Agilent Technologies, Massy, France). Only patients with a RNA Integrity number greater than 7 (determined by the Bioanalyzer) and a 28S/18S value greater than 1 were selected for the following studies.
|
| Label |
biotin
|
| Label protocol |
250 ng of total RNA was amplified using TotalPrep RNA Amplification kit (Ambion, Applied Biosystems, Courtaboeuf, France) and biotin-16-UTP were incorporated. Amplified RNA concentration and quality were determined using Nanodrop and Agilent 2100 Bioanalyzer (RNA nano LabChip). 750 ng of amplified RNA were used for DirectHyb Assay where RNA is labelled with Streptavidin-Cy3 following illumina's protocol.
|
| |
|
| Hybridization protocol |
750 ng of amplified and Cy3-labelled RNA were used directly hybridized on the Beadchips for 16 hours at 58°C following Illumina’s instructions. Beadchips were then washed following Illumina's protocol.
|
| Scan protocol |
Beadchips were scanned on the Illumina BeadXpress® platform available on the site of hospital La Pitié-Salpêtrière (http://www.p3s.chups.jussieu.fr/) according to the manufacturer’s protocol.
|
| Description |
The array was first analyzed using Illumina's BeadScan software. The array was then checked for background and hybridisation intensities and homogeneity.
|
| Data processing |
Raw data were normalized with the Quantiles-method using IlluminaGUI R-package (Schultze and Eggle, Bioinformatics 2007 23(11):1431-1433). Present/Absent call was determined with the detection p-values using IlluminaGUI. Analysis (comparisons between patients under 60 named young and patients over 75 named old) was then performed on the 13792 probes which are called Present in at least half of the samples of one of the 2 groups (young or old).
|
| |
|
| Submission date |
Jun 08, 2009 |
| Last update date |
Dec 07, 2010 |
| Contact name |
Sophie Nadaud |
| E-mail(s) |
[email protected]
|
| Phone |
33 1 40779686
|
| Fax |
33 1 40779645
|
| Organization name |
INSERM
|
| Department |
U956
|
| Street address |
91 Bd de l'hopital
|
| City |
Paris |
| ZIP/Postal code |
75013 |
| Country |
France |
| |
|
| Platform ID |
GPL6104 |
| Series (1) |
| GSE16487 |
Gene expression profiling of human mammary artery media during aging |
|
